Novel condensation products having high activity to insolubilize proteins and protein-insolubilized products

ABSTRACT

The novel condensation product of 1,3-phenylenediamine and glutardialdehyde has high activity to fix or insolubilize proteins, including enzymes.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Pat. applications,Ser. No. 440,438, filed Feb. 7, 1974, now abandoned and Ser. No.571,187, filed Apr. 24, 1975, now abandoned. The latter application is adivisional application of Ser. No. 440,438. This application is alsorelated to Ser. No. 593,605, filed July 7, 1975, now abandoned, and toco-pending Ser. No. 714,153, filed Aug. 13, 1976, now U.S. Pat. No.4,066,504 which is a division of Ser. No. 593,605 abandoned.

FIELD OF THE INVENTION

This invention relates to the field of insolubilizing proteins.

The relationship of this application to the first two aforesaidapplications is that this is a continuation-in-part application of thefirst two applications mentioned. A clear line of distinction exists asto the claims in all three applications.

PRIOR ART

The manufacture of a condensation resin having a large bonding capacityfor proteins, by the reaction of 1,3-phenylenediamine and formaldehyde,as well as diazotisation of the reaction product, is known.

It has surprisingly been found, in accordance with the presentinvention, that a substantially more active product with respect to thefixing or insolubilizing of proteins is obtained when the novelcondensation products of the present invention are used as carriers forthe proteins.

Co-pending divisional applications of this application are Ser. Nos.821,367, 821,362 and 821,663 all filed Aug. 3, 1977.

SUMMARY OF THE INVENTION

It is known that proteins, especially enzymes, can be bonded to solidcarrier materials to give insoluble products which have advantageousproperties. The bonded, insolubilized, but biologially active, proteinscan, for example, be more readily handled as in their naturally solubleform. Because they can be readily recovered, enzymes bonded to a carriercan be used several times in a stationary process or, with particularadvantage, in a process which is continuously worked.

Thus, for example, the manufacture of a condensation resin having alarge bonding capacity for proteins by the reaction of1,3-phenylenediamine and formaldehyde as well as the diazotisation ofthe reaction product are known.

It has now surprisingly been found in accordance with the presentinvention that a substantially more active product with respect to thefixing or insolubilizing of proteins is obtained when1,3-phenylenediamine is condensed with glutardialdehyde.

The present invention is based on the foregoing finding and isaccordingly concerned with a novel condensation product manufactured bythe condensation of 1,3-phenylenediamine with glutardialdehyde.

As compared with the aforementioned known condensation productmanufactured from 1,3-phenylenediamine and formaldehyde, the novelcondensation product manufactured from 1,3-phenylenediamine andglutardialdehyde has a number of very advantageous properties which makeit excellently suitable as a carrier material for the fixing of proteinsof the most diverse type and origin: it forms larger particles which areeasier to filter (i.e. free flow is guaranteed when the material isplaced on a G-1 to G-4 sintered glass disc. No suction or pressure isrequired), quicker to sediment and therefore generally better to handle;the bonding capacity for proteins is substantially greater; the novelcondensation product is already an activated carrier which can be usedimmediately and can bring about the fixing of the protein in a verysimple manner because of the high activity. Finally, the condensationproduct of this invention is very inexpensive.

The invention is also concerned with a process for the manufacture ofthe aforementioned novel condensation product, namely1,3-phenylenediamine glutardialdehyde resin (PAG-resin), which processcomprises reacting 1,3-phenylenediamine with glutardialdehyde. Theinvention is further concerned with a process for the fixing of proteinsto the PAG-resin and with the proteins fixed to the PAG-resin.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Proteins which can be bonded to the condensation products in accordancewith the present invention include polypeptides, antigens, antibodies,protein inhibitors and, especially, enzymes. The enzymes can be ofvegetable, animal or microbial origin. The enzymes may be hydrolases(peptidases, proteinases, desaminases, carbohydrases, esterases,nucleases), lyases or desmolases (hydrolyases, decarboxylases,aldolases), transferases, isomerases, oxidoreductases and ligases.Examples of enzymes from which insoluble enzyme preparations provided bythis invention can be manufactured are alcoholdehydrogenase,naringinase, hesperidinase, β-glucosidase, α-amylase, invertase,amyloglucosidase, urease trypsin, ficin, papain, bromelin,subtilopeptidase, rennin, glucoseisomerase, glucoseoxidase, peroxidase,catalase, acylase, cytochrome, ribonuclase, phosphodiesterase andadenyldeaminase.

The molar ratio of the polyamine and aldehyde reaction productsexpediently lies at between 1:1 and 1:10, a ratio of about 1:3 beingpreferred.

The reaction can be carried out in a manner known per se; for example,in aqueous solution, preferably with the addition of an acid such as amineral acid (e.g. hydrochloric acid). In a particular embodiment, thereaction is carried out in the presence of an inert, fine-grained,preferably inorganic, especially silicate-containing, adjuvant. Examplesof such adjuvants are silica gel, pumice-stone, diatomaceous earth(kieselguhr), bentonite, woolastonite, porous glass and also metaloxides such as aluminium oxide or hydroxylapatite. It is preferred touse silica gel (e.g. with a particle size of 0.05-0.2 mm, 70-325 mesh)or pumice stone (e.g. with a particle size of 0.05-10 mm). The presenceof such an adjuvant gives rise to a homogeneous particle formation inthe reaction and, as a result an improved sedimentation is achieved. Thereaction in the presence of an adjuvant is expediently carried out byinitially bringing the particles into contact with one of the tworeaction components and then adding the second component withsimultaneous or subsequent slight acidification.

The reaction can be carried out in a homogeneous phase, or, preferably,in a two-phase system with the addition of an acid such as a mineralacid (e.g. sulphuric acid, phosphoric acid or, preferably, hydrochloricacid) or an organic acid (e.g. a carboxylic acid such as acetic acid)with vigorous stirring or shaking. The use of a two-phase systempromotes the formation of spherical, substantially homogeneous particleswhich are easy to filter and which sediment well, these particles beingespecially well suited as a carrier material. The reaction products areamorphous materials which are insoluble in water and the usual organicsolvents. The exact structure of the polymer is not known, however,quinoline, 1,7-phenanthroline and 1,6-diazaphenalene units are presentin the molecules.

Inert, water-immiscible organic solvents (e.g. dichloromethane,chloroform, carbon tetrachloride, benzene, toluene, ethyl acetate,dioxane, carbon disulphide) are suitable as the second phase. Chloroformis preferably used as the inert, water-immiscible solvent, but acetoneis also suitable for this purpose.

The reaction temperature is not critical; it can lie, for example, atbetween 0° C. and 50° C., preferably at room temperature (i.e. at about18°-22° C.).

In accordance with the process provided by this invention there isobtained a condensation product carrier material which, although alreadyvery active, can be still further activated by diazotisation. Thediazotisation can be carried out in a manner known per se by treatmentwith nitrite and acid.

In order to fix the protein to the carrier material, the latter istreated with an aqueous solution, preferably a buffered solution, of theprotein (1-50 mg/ml) at a temperature of 0°-30° C., preferably 4° C. toroom temperature. This treatment can be carried out while stirring orshaking. Because of the high activity of the carrier material, thefixing of the proteins can also be advantageously carried out by simplyfiltering the protein solution through a carrier layer, preferably acolumn filled with the carrier material, as is common, for example, incolumn chromatography. Thus, for example, culture filtrates ofmicroorganisms which contain proteins or enzymes can be allowed to rundirectly through a column of carrier material, the proteins or enzymesbeing selectively fixed to the carrier. The column is expediently rinsedwith a buffer solution and 1-M potassium chloride solution in order toremove the non-fixed proteins.

The proteins fixed to the carrier material are generally very stable andvery high specific activities (unit/g) are achieved in the case ofenzymes. Loadings of, for example, 1:3-10 parts by weight ofprotein/carrier material are achieved.

The carrier-bonded proteins provided by this invention, especiallyenzymes, can be used in a manner known per se; for example, foranalytical or preparative purposes or in food technology such as in themanufacture of glucose from starch [see, for example, ScientificAmerican 224, (No. 3) 26-33 (1971); Angew. Chemie 84, (8) 319-268(1972); Chemiker Zeitung 96, (11), 595-602 (1972); C & EN, (15.2.71),86-87].

In this important large-scale process, amyloglucosidase fixed to thecondensation product provided by the present invention can be utilisedfor the manufacture of glucose from the "glucose syrup" (prehydrolysedstarch). The condensation products can then be transferred, for example,onto columns (fixed-bed process). A further application results in theenzymatic degradation of lactose in milk products by means of lactases(e.g. "sweets" from whey).

The following Examples illustrate the present invention. Thequantitative data of the carrier material obtained or used is given withrespect to dry weight.

EXAMPLE 1

50 g of glutardialdehyde (25% aqueous solution) were added with vigorousstirring to a solution of 5.0 g of 1,3-phenylenediamine in 50 ml ofwater and 10 ml of concentrated hydrochloric acid. When solidificationof the reaction mixture occurred, 300 ml of water were added. Themixture was stirred for a further hour at room temperature and thenevaporated under reduced pressure via a Buchner funnel. The finepolymeric particles were washed with 0.1-N sodium hydroxide and waterand stored in 0.01-N sodium hydroxide. The yield amounted to 13.8 g.

EXAMPLE 2

50 g of glutardialdehyde were added with vigorous stirring to a solutionof 5 g of 1,3-phenylenediamine in 300 ml of chloroform and, after afurther 5 minutes, 20 ml of 7-N hydrochloric acid were added. Thereaction mixture solidified immediately. It was treated with 100 ml ofwater and shaken for 5 minutes until it again became liquid. Afterfiltration of the mixture under a vacuum via a Buchner funnel, thepolymeric particles were washed with 0.1-N sodium hydroxide. Theresidual chloroform was removed by washing with acetone. The yieldamounted to 14 g. The thus-obtained carrier particles were spherical,homogeneous and capable of good filtration. They were stored in water orin dilute sodium hydroxide.

EXAMPLE 3

30 g of silica gel were added with stirring to a solution of 2.0 g of1,3-phenylenediamine in 50 ml of benzene. After stirring for 10 minutesat room temperature, the silica gel was filtered off via a Buchnerfunnel and sucked dry. Benzene still adhering to the silica gel did notinterfere with the further reaction. The silica gel particlesimpregnated with 1,3-phenylenediamine were then rapidly added withvigorous stirring to a mixture of 50 ml of glutardialdehyde (25%solution in water), 10 ml of concentrated hydrochloric acid and 340 mlof water. Polymerisation occurred immediately. The orange-red polymerwas stirred for 1 hour at room temperature, filtered off under reducedpressure via a Buchner funnel and washed with water until neutral. Thethus-obtained carrier material was then stored in a weakly alkalinesolution (0.01-N sodium hydroxide).

EXAMPLE 4

30 g of silica gel were added with vigorous stirring to 50 g ofglutardialdehyde (25% aqueous solution). The mass was then added withvigorous shaking to a solution of 5 g of 1,3-phenylenediamine in 300 mlof chloroform. After the addition of 10 ml of 7-N hydrochloric acid and100 ml of water, the mixture was shaken for a further 10 minutes. Theproduct was worked up in the same manner as described in Example 2.There were obtained homogeneous, spherical particles which sedimentedquickly and which could be filtered in an excellent manner.

EXAMPLE 5

Carrier materials were manufactured in a manner analogous to thatdescribed in Example 3 and Example 4 using diatomaceous earth in placeof silica gel.

EXAMPLE 6

A suspension of 1 g of the condensation product obtained according toExamples 1-5 in 30 ml of water was acidified with 1.15 ml ofconcentrated hydrochloric acid at 0° C. A 1-N sodium nitrite solutionwas added dropwise, with stirring, to the ice-cold suspension. At theend of the diazotisation, the black-violet coloured carrier material wasfiltered off, washed with cold water and a buffer solution (pH 4-8.5,depending on the pH value of the protein solution which is subsequentlyto be fixed to the carrier material).

EXAMPLE 7

0.23 g of the carrier material obtained according to Example 2 werediazotised in the manner described in Example 6 and filled into achromatography column (1.5×15 cm). A solution of 1 g of amyloglucosidase(52 units/mg) in 100 ml of 16 mmol acetate buffer (pH 4.8) was passedthrough the column at room temperature and with a flow rate of 20 ml perhour. The fixed enzyme had an activity of 7,000 units/g of carriermaterial.

EXAMPLE 8

A suspension of 1 g of diazotised carrier material (obtained accordingto Example 3 or Example 4) in 20 mmol of phosphate buffer (pH 5.2) wasfilled into a chromatography column (1.5×15 cm). A solution of 500 mg ofinvertase (150 units/mg) in 50 ml of 20 mmol phosphate buffer (pH 5.2)was passed through the column at room temperature and with a flow rateof 20 ml per hour. The column was washed with 50 ml of 20 mmol phosphatebuffer (pH 5.2) and 50 ml of 1-N potassium chloride solution. The amountof fixed enzyme was 349 mg. The activity of the fixed enzyme was 4,280units/g of carrier material. The activity of the bonded enzyme was 8.5%compared with the soluble enzyme. The weight ratio of enzyme to carriermaterial was about 1:3. An invertase preparation manufactured in ananalogous manner from 20 g of diazotised carrier material showed anactivity of 17.6% compared with that of the soluble enzyme.

EXAMPLE 9

1 g of diazotised carrier material (manufactured according to Example 3or Example 4) in 0.1-M phosphate buffer (pH 7.0) was filled into achromatography column (1.5×15 cm). A solution of 600 mg of ficin in 60ml of a solution of 0.1-M phosphate buffer, 7 mmol of mercaptoethanoland 1 mmol of EDTA (pH 7.0) was passed through the column at roomtemperature and with a flow rate of 20 ml per hour. The column wasrinsed with 50 ml of the same buffer solution and 50 ml of 1-M potassiumchloride solution. The amount of fixed enzyme was 459 mg. The activityof the fixed enzyme determined with casein as the substrate amounted to15% of the activity of the soluble enzyme.

EXAMPLE 10

A solution of 4 g of naringinase in 100 ml of 0.05-M citrate buffer (pH6.5) was passed at room temperature and with a flow rate of 20 ml perhour through a column containing 2 g of diazotised carrier materialobtained according to Example 3 or Example 4. The carrier-bondednaringinase showed an activity of 32% compared with the soluble enzyme.

EXAMPLE 11

2 g of diazotised carrier material obtained according to Example 3 orExample 4 were shaken for 1 hour at 4° C. with a solution of 600 mg ofnaringinase in 50 ml of 0.05-M citrate buffer (pH 6.5). The activity ofthe fixed naringinase amounted to 50% compared with that of the solubleenzyme.

EXAMPLE 12

In an analogous manner as described in Example 11, 40 mg ofhesperidinase in 50 ml of 0.05-M citrate buffer (pH 6.5) were bonded tothe carrier material. The activity of the fixed enzyme amounted to 27%of that of the soluble enzyme.

EXAMPLE 13

A solution of unpurified glucose-isomerase, obtained from the partiallypurified culture filtrate of a microorganism (Streptomyces glaucescens),was passed through a column containing 50 g of diazotised carriermaterial manufactured according to Example 3 or Example 4. The columnwas washed with 0.05-M phosphate buffer (pH 6.5) and 1-M potassiumchloride solution. The activity of the fixed glucose-isomerase amountedto 74% of that of the soluble enzyme, determined by measuring theisomerisation of a 10% glucose solution of fructose.

EXAMPLE 14

A solution of 100 mg of bovine serum albumin in 50 ml of 0.05-Mphosphate buffer (pH 6.5) was passed through a chromatography column(1.5×5 cm) containing 0.5 g of diazotised carrier material, obtainedaccording to Example 3 or Example 4, at room temperature and at a flowrate of 20 ml per hour. 68.4 mg of the albumin was bonded to the carrierso that the weight ratio of bonded protein to carrier material amountedto about 1.4:10.

Approximately the same coupling ratio was obtained when a non-diazotisedcarrier material was used.

What we claim is:
 1. A process for the preparation of a condensationproduct, which process comprises reacting 1,3-phenylene-diamine withglutardialdehyde to form a product insoluble in water and inert,water-immiscible organic solvents, and diazotizing said product.
 2. Thediazotized product obtained in accordance with claim 1.